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Comparative study of non-autolytic mutant and wildtype strains of Coprinopsis cinerea supports an important role of glucanases in fruiting body autolysis Zhonghua Liu, Xin Niu, Jun Wang, Wenming Zhang, Mingmei Yang, Cuicui Liu, Yuanjing Xiong, Yan Zhao, Siyu Pei, Qin Qin, Yu Zhang, Yuan Yu, and Sheng Yuan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03962 • Publication Date (Web): 09 Oct 2015 Downloaded from http://pubs.acs.org on October 12, 2015
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Journal of Agricultural and Food Chemistry
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Comparative study of non-autolytic mutant and wild-type strains of Coprinopsis
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cinerea supports an important role of glucanases in fruiting body autolysis
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Zhonghua Liu, Xin Niu, Jun Wang, Wenming Zhang, Mingmei Yang, Cuicui Liu,
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Yuanjing Xiong, Yan Zhao, Siyu Pei, Qin Qin, Yu Zhang, Yuan Yu and Sheng
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Yuan*.
7 8
*Corresponding author:
[email protected] 9 10
Sheng Yuan
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College of Life Science
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Nanjing Normal University
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1 Wenyuan Rd
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Xianlin University Park
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Nanjing, 210023
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PR China
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Tel: 86-25-85891067 (O)
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Fax: 86-25-85891067 (O)
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Abstract
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Autolysis of Coprinopsis cinerea fruiting bodies affects its commercial value. In this
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study, a mutant of C. cinerea that exhibits pileus expansion without pileus autolysis
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was obtained using ultraviolet mutagenesis. This suggests that pileus expansion and
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pileus autolysis involve different enzymes or proteins. Among the detected hydrolytic
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enzymes, only β-1,3-glucanase activity increased with expansion and autolysis of
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pilei in the wild-type strain, but the increase was abolished in the mutant. This
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suggests that β-1,3-glucanases plays a major role in the autolysis. Although there are
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forty-three possible β-1,3-glucoside hydrolases genes, only four known genes, which
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have products that are thought to act synergistically to degrade the β-1,3-glucan
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backbone of cell walls during fruiting body autolysis, and an unreported gene were
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up-regulated during pileus expansion and autolysis in the wild-type stain, but were
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suppressed in the mutant. This suggests that expression of these β-1,3-glucanases are
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potentially controlled by a single regulatory mechanism.
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Key Words: Coprinopsis cinerea, fruiting body, pileus expansion, pileus autolysis,
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β-1,3-glucoside hydrolase
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Introduction
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Edible mushrooms are often prone to autolysis postharvest, resulting in a short shelf
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life that affects the commodity value and sale of the mushrooms. For example, straw
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mushroom (Volvaria volvacea) fruiting bodies undergo rapid autolysis and
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liquefaction postharvest 1. Although other mushroom fruiting bodies are generally
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unlikely to lyse quickly and liquefy during maturation, the autolysis process still
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occurs. An example is the browning phenomenon that occurs in shiitake mushroom
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(Lentinus edodes) fruiting bodies postharvest, which is accompanied by the hydrolysis
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of an active ingredient, lentinan 2,3. Therefore, autolysis of mushrooms severely limits
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commercialization, shortens the shelf life, substantially increases the difficulty and
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cost of storage, and negatively impacts the preservation process for edible mushrooms.
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Coprinopsis cinerea (also known as Coprinus cinereus) is a model mushroom that is
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associated with strong fruiting body autolysis. The pileus of C. cinerea expands and
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autolysis quickly occurs upon the maturity of the fruiting bodies. Ultimately, when the
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pileus completely liquefies, an inky liquid is formed and released. Moreover, it is easy
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to induce mutations in C. cinerea, and its clear genetic background facilitates genetic
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manipulation and gene analysis. Therefore, C. cinerea is ideal for studying autolysis
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in mushrooms postharvest 4.
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The aim of this study is to create a non-autolysis mutant by using ultraviolet
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mutagenesis to extend the shelf life of C. cinerea mushrooms and improve their
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commercial value. The study reports an identification of a non-autolytic C. cinerea
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mutant. Surprisingly, a comparative study of a non-autolytic mutant and the wild-type 3
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strain of C. cinerea demonstrated that β-1,3-glucanases rather than chitinase play a
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major role in the autolysis of fruiting bodies. Furthermore, only five genes among the
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forty-three possible β-1,3-glucoside hydrolases genes detected were associated with
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autolysis of fruiting bodies.
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Materials and methods
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Strains and culture conditions
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C. cinerea ATCC 56838 was purchased from American type culture collection
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(ATCC). The culture of fruiting bodies of the wild-type and mutant strains was
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described by Zhang et al. 5. After the stipe of C. cinerea ceased elongation (8-10 cm
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long), the photographs were taken when the pileus of wild-type and mutant strains
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expand to different degrees (0°, 90° and 180°).
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Ultraviolet mutagenesis
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The non-autolytic mutant strain was obtained via ultraviolet mutagenesis according to
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Muraguchi et al. 6. To obtain the oidia of the C. cinerea, four agar blocks with
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mycelium was inoculated on the yeast malt glucose (YMG) medium agar (4 g yeast
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extract, 4 g glucose, 10g malt extract and 1 l distilled water) in Petri dishes (9 cm in
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diameter) and incubated at 37 °C in darkness for 5 days. Then the oidia in the Petri
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dishes were suspended in 5 ml sterile water by scraping with a sterile blunt spatula,
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and purified by filtration through glass wool. One ml of oidia suspension in a 9 cm
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Petri dish was irradiated by a CL-1000 Ultraviolet Crosslinker (Upland, CA, USA) 4
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with an irradiation energy of 200 µJ/cm2. The UV-irradiated oidiospores were
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uniformly spread on MM + AA medium and incubated in darkness. The single
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colonies that formed were transferred to YMG medium and cultured using the same
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method as the wild-type strain.
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Pileus hydrolase extraction
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The degree of pileus expansion was observed when the stipe of C. cinerea elongated
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to 8–10 cm in length and ceased elongation. Pileus tissue samples were collected from
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the fruiting bodies of wild-type and mutant strains at different degrees (0°, 90° and
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180°) of pileus expansion. The samples were ground in liquid nitrogen to form a fine
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powder and then added to two volumes of NaAc-HAc buffer (50 mM, pH 6.0). The
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mixture was fully shaken and centrifuged at 12,000 g for 10 min. The supernatant was
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collected and dialyzed in NaAc-HAc buffer (50 mM, pH 6.0) to remove reducing
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sugars and other substances in the extract that could affect enzyme activity assays.
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Following dialysis, the crude enzyme solution was centrifuged at 12,000 g for 10 min,
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and the supernatant was placed on ice before use.
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Hydrolase activity assays
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To analyze glucanase and chitinase activities in crude pileus extracts, 100 µL of 1%
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laminarin (β-1,3-1,6-glucan) (L9634, Sigma) or 1% chitin (C7170, Sigma) was
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thoroughly mixed with 100 µL of 50 mM NaAc-HAc (pH 6.0) containing appropriate
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amounts of enzymes and incubated at 37°C and 800 rpm for 20 min for glucanase or 5
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60 min for chitinase. After incubation, 200 µL of DNS reagent was added to the
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reaction, followed by incubation at 100°C for 10 min. The reaction was then placed
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on ice for 2 min. After centrifugation, the absorbance of the supernatant at OD520
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was measured 7. One unit of enzyme activity was defined as the amount of enzyme
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that released 1.0 mol of reducing sugar (glucose standard) per min under the assay
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conditions.
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To analyze peptidase activity in crude pileus extracts, 100 µL of 1% casein (CN5204,
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BBI) was thoroughly mixed with 100 µL of 50 mM NaAc-HAc (pH 6.0) containing
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appropriate amounts of enzymes, followed by incubation at 37°C and 800 rpm for 30
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min. At the end of the reaction, the concentration of free tyrosine in the hydrolysate
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was measured using a Folin-Phenol Reagent Kit (SK5031, BBI). One unit of enzyme
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activity was defined as the amount of enzyme that released 1.0 mol of tyrosine per
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min under the assay conditions.
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RNA extraction and quantitative PCR
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Total RNA was extracted from the pileus tissue of the wild-type and mutant strains
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using a Spin Column Fungal Total RNA Purification Kit (SK8659, BBI). Removal of
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residual genomic DNA and transcription and synthesis of cDNA was performed using
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a HiScript® II Q RT SuperMix for qPCR kit (+gDNA wiper) (R223-01, Vazyme).
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Quantitative PCR was performed using amplification mixtures containing TaKaRa
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SYBR Premix Ex Taq II (Tli RNase H Plus) (TaKaRa), reverse-transcribed RNA and
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primers. β-Tubulin was used to standardize the mRNA levels. Reactions were run on a 6
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StepOnePlusTM Real Time PCR System (Applied Biosystems). Cycle threshold (CT)
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values were used to calculate the mean fold changes of the reactions via the 2−∆∆CT
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method. −∆∆CT = (CT.Target-CT.β-tubulin) 90° / 180°-(CT.Target-CT.β-tubulin) 0° (Livak
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and Schmittgen, 2001). The primers used for qPCR are listed in the supplemental
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documents.
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Results
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Comparison of the mutant and wild-type strain fruiting body morphologies
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Autolysis postharvest of basidiomycete fruiting bodies occurs with the action of a
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series of hydrolytic enzymes, so the disruption of a single hydrolase gene is not
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effective in suppressing the autolysis of fruiting bodies 8. The intent of this study is to
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create non-autolysis mutants by ultraviolent mutagenesis to identify the hydrolytic
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enzymes involved in autolysis postharvest.
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Among the non-autolysis mutants, we found an interesting mutant that exhibited the
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following two remarkable characteristics: (1) it produces less black basidiospores than
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the wild-type strain, causing the pilei to appear white, (2) the pilei continue to expand
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but lose autolysis at maturation, i.e., there is a defect in the collapse and liquefaction
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of the pileus. As shown in Fig. 1, when the stipe of C. cinerea ceased elongation at
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8-10 cm, the pileus remained unexpanded (0°) (Fig. 1.1). Approximately 6 h later, the
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pileus expanded to approximately 90° (Fig. 1.2). After the stipe ceased elongation for
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12 h, the pileus fully expanded to 180° (Fig. 1.3). At approximately 12 h after full
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expansion of the pileus, the pileus completely collapsed and liquefied (Fig. 1A4). 7
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With the exception of the last stage, in the above first three stages, the morphology of
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the pileus was not different between the mutant and wild-type stains of C. cinerea, but
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the color of the pileus appeared to be significantly different. For the wild-type strain,
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the massive production of mature black basidiospores resulted in a black color to the
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pileus (Fig. 1, A1-A3). For the mutant at the same stage, there was little production of
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basidiospores, therefore, the pileus appeared white (Fig. 1, B1-B3). Differential
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interference contrast (DIC) microscopy revealed that although a large number of
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black basidiospores was retained in the pileus residues of the wild-type fruiting body,
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no intact cell structure was observed (Fig. 2A). For the mutant, the fruiting body did
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not lyse to cause the collapse and liquefaction of the pileus at 12 h following full
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pileus expansion and maintained an intact umbrella-like structure (Fig. 1B4). DIC
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observation revealed that only a small number of black basidiospores was present in
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the pileus of the mutant, with a fully intact cell structure (Fig. 2B). The above results
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indicated that autolysis of the fruiting body was inhibited at maturity in the mutant.
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Comparison of glucanase, chitinase and peptidase activities during the pileus
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expansion and autolysis of the fruiting body of mutant and wild-type strains
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A hydrolytic enzyme analysis (Fig. 3A) showed that the glucanase activity in the
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pileus at no expansion (0°) was 3.74 U/mg of fresh weight for the wild-type strain.
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The enzyme activity increased rapidly to 11.65 U/mg of fresh weight in the pileus at
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half expansion (90°), demonstrating a 3.12-fold increase. At full expansion (180°),
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glucanase activity occurred at 9.58 U/mg of fresh weight, which represented a 8
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2.56-fold increase relative to the activity at no expansion and a 17.77% decrease
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relative to the activity at half expansion. This slight decrease might be related to the
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inactivation or degradation of some glucanase in the late stage of pileus autolysis.
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However, for the mutant, although the glucanase activity in the pileus at no expansion
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was not significantly different from the wild stain (P > 0.05), the glucanase activity in
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the pileus was strongly suppressed and did not increase during pileus expansion and
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autolysis, in contrast to the wild-type strain in which glucanase activity increased.
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However, the chitinase activity in the pileus followed a different trend than the
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glucanase activity (Fig. 3B). In the wild-type strain, the chitinase activity was 0.16
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U/mg of fresh weight in the pileus with no expansion and increased rapidly to 0.71
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U/mg of fresh weight in the pileus at half expansion, demonstrating a 4.44-fold
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increase. At full expansion of the pileus, the chitinase activity was 0.56 U/mg of fresh
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weight, which represented a decrease compared to the pileus at half expansion, but
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was not significantly different (P > 0.05). Similarly, for the mutant, the chitinase
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activity in the pileus with no expansion was 0.30 U/mg of fresh weight, which was
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not significantly different from the wild strain (P > 0.05). The chitinase activity in the
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pileus at half expansion and at full expansion increased to 0.66 U/mg of fresh weight
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and 0.64 U/mg of fresh weight, respectively. Thus, no significant differences in
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chitinase activity were observed in the pileus of the mutant compared to the wild-type
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strain at the three stages (P > 0.05), i.e., the chitinase activity in the pileus was not
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apparently reduced in the non-autolysis mutant during pileus expansion compared to
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wild strain. 9
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The peptidase activity in the pileus of C. cinerea followed different trends compared
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to glucanase and chitinase activities (Fig. 3C). For the wild-type strain, the peptidase
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activity in the pileus at no, half and full expansion occurred at 1.44, 1.45 and 1.62
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U/mg of fresh weight, respectively, demonstrating no significant increase with pileus
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expansion and autolysis (P > 0.05). For the mutant, the peptidase activity in the pileus
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during the three stages occurred at 1.32, 1.29 and 1.45 U/mg of fresh weight. There
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were no significant differences in peptidase activity in the mutant between different
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stages (P > 0.05) or compared to the wild-type strain (P > 0.05).
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In summary, the above results indicated that for the mutant strain, a significant
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reduction in glucanase activity may contribute to the inhibition of autolysis of the
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pileus.
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Comparison of the glucanase expression patterns between the mutant and
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wild-type strains during fruiting body maturation
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Whole-genome sequencing of C. cinerea okayama7 #130 has been completed. The
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analysis
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genome/?term=Coprinopsis%20cinerea) revealed that sixty-four proteins were
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annotated as glucanases in the okayama7 #130 genome. These proteins were analyzed
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using
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Structure/cdd/wrpsb.cgi?). The results showed that among sixty-four proteins, there
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are two proteins from the glycosyl hydrolase family (GHF) 1, fifteen proteins from
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GHF 2, five proteins from GHF 3, three proteins from GHF 5, fifteen proteins from
of
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genomic
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(http://www.ncbi.nlm.nih.gov/
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GHF 16, two proteins from GHF 17, sixteen proteins from GHF 61, and one protein
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each from GHF 6, 9, 15, 55, 63 and 88 (Supple Table 1). Because five proteins
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belonging to GHF 1, 6, 15 and 88 mainly act on β-1,4-glucan bonds and the sixteen
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proteins belonging to GHF 61 usually lack measurable glucanase activity on their own,
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but enhance the activity of other cellulolytic enzymes 9-11, we used a qPCR technique
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to assay the expression levels of the remaining forty-three GHF proteins in the
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wild-type strain pileus. The experimental results showed that among the transcribed
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forty-three genes, the following five glucanase genes were expressed at remarkably
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higher levels than the others: EAU92553 of GHF 2, EAU87688 of GHF 3,
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EAU84955 of GHF 16, EAU85332 of GHF 17 and EAU86655 of GHF 55 (Fig. 4).
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Interestingly, EAU92553 of GHF 2, EAU84955 of GHF 16, and EAU86655 of GHF
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55 are, respectively, a β-1,3-glucosidase BGL1, an endo-β-1,3-glucanase ENG, and
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an exo-β-1,3-glucanase EXG from C. cinerea pilei, previously reported 12, EAU87688
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of GHF 3 is an extracellular β-glucosidase from the C. cinerea pileus and stipe
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and EAU85332 of GHF 17 is a previously unreported enzyme, annotated as a putative
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glucan 1,3-β-glucosidase in the NCBI.
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We further assayed and compared the change in RNA levels of the five
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high-expression glucanases during pileus autolysis in the wild-type and mutant strains.
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The transcript levels of the following five glucanases in the pileus were measured at
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different stages: the no expansion (0°), the half expansion (90°), and the full
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expansion (180°) of pileus. As shown in Fig 5, compared to the unexpanded pileus,
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the transcript levels of all of the five glucanases were upregulated in the pileus of 11
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mature fruiting bodies of wild type stain at the half expansion stage (90°) in the
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wild-type strain. The mRNA level of the five glucanases increased more than 2-fold.
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At full expansion stage (180°), the expression levels of the five glucanases decreased
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compared to the half expansion state. In contrast, the transcription of these five
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glucanase genes were suppressed in the pileus of mature fruiting bodies of the mutant
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strain. In the unexpanded pileus and the half expanded pileus, the mRNA level of the
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five glucanases decreased by over 95% in the mutant strain, compare to the wild-type
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strain. It needs to mention that nearly half of the remaining glucanases among the
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detected forty-three GHF proteins were randomly selected to test their expression
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levels at the three different stages. The results showed that the remaining glucanases
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was not expressed or at a low level in the pileus of mature fruiting bodies (data not
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show).
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Discussion
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C. cinerea forms a large number of black basidiospores in the pileus after fruiting
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body maturation, rendering the fruiting bodies black. Once matured, basidiospores are
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released by C. cinerea through pileus expansion and autolysis4. A series of mutants
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with varying defects have been obtained by ultraviolet mutagenesis of C. cinerea
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oidiospores. There are two main types of mutants associated with the pileus. One type
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has a white pileus during autolysis because it is unable to produce black basidiospores
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and thus appears white after fruiting body maturation6
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non-expanding pileus with basidiospores4. Via ultraviolet mutagenesis, Muraguchi et 12
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; the other type has a
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al.4 screened a non-autolytic strain with a non-expanding pileus. The authors
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concluded that the processes of pileus expansion and autolysis are coupled in C.
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cinerea: basidiospores are released from the pileus and dispersed as the pileus
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expands and autolysis, and the basidiospores that are not released from the pileus fall
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down as inky drops when the pileus collapse and liquefies4. In the present study, we
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performed ultraviolet mutagenesis of oidiospores to obtain a mutant form of C.
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cinerea with a white pileus and pileus expansion that does not collapse and liquefy.
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To our knowledge, this mutant is the first C. cinerea strain reported to reach full
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pileus expansion without liquefaction. We propose that pileus expansion and pileus
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autolysis may involve different enzymes or proteins, respectively. We previously
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reported that one expansin-like protein from snail stomach juice could reconstitute
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heat-inactivated stipe wall extensions in C. cinerea stipes without hydrolytic activity15.
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It would be interesting to determine if some similar expansin-like proteins mediate the
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pileus expansion process in a non-lytic way in the future.
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Autolysis of C. cinerea fruiting bodies is a degradation process of the cell wall. It is
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known that the fungal cell wall is primarily composed of β-1,3-glucan with
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β-1,6-linked branches, chitin and proteins16. It has been suggested that some
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glucanases, chitinases and peptidases are involved in C. cinerea pileus expansion and
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autolysis17-20, or in enescence (a result of cell wall degradation) of basidiomycete
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Lentinula edodes fruiting bodies after harvesting21, or the autolysis of hyphal cell wall
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degradation in ascomycete Aspergillus nidulans initiated by carbon starvation22-26.
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This study shows that glucanase and chitinase activities are elevated in the pileus 13
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during fruiting body maturation and that both peaked at half expansion of the pileus
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(90°), whereas the peptidase activity remained nearly unchanged throughout the
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process of pileus expansion and autolysis. Therefore, an involvement of the peptidase
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in C. cinerea pileus autolysis and liquefaction during fruiting body maturation may be
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excluded.
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Iten et al.17 earlier reported that chitinases were newly formed shortly before spore
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release begins in fruiting bodies of Coprinus lagopus. Therefore, they suggested that
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pileus autolysis is accomplished by the action of chitinase. However, Iten et al.17
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ignored the fact that glucanase activity was also quickly increased before spore
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release began probably because glucanases were present at a lower amount in fruiting
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bodies. Later, Bush et al.18 tested the activity of the glucanases in the mature fruiting
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bodies of Coprinus comatus and found that there appeared to be approximately 26
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times as much β-1,3-glucanase as chitinase in the 24 h autolysate of fruiting bodies.
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They suggested that β-1,3-glucanase plays a major role in the autolysis of fruiting
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bodies. However, by qPCR analysis, Lim et al.19 and Kang et al.20 recently found that
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the expression of two chitinases were increased in Coprinellus congregatus at the
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mature mushroom stage compared to primordia or young mushrooms, so they
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proposed that chitinase is involved in the degradation of mushroom cell walls during
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the autolysis that generates the black liquid droplets. This study showed that chitinase
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activity was indeed increased when fruiting bodies entered the maturation stage
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compared to young fruiting bodies; however, this increase in chitinases activity during
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the maturation of fruiting bodies was not changed in the non-autolysis mutant of C. 14
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cinerea. In contrast, glucanase activity no longer increased with fruiting body
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maturation in the pileus of the non-autolytic mutant strain compared to WT strain and
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was almost unchanged during maturation of fruiting bodies of the non-autolytic
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mutant strain. This is the first convincing evidence for a major role of glucanases in
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the autolysis of fruiting bodies. Certainly, this study does not rule out chitinases
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involving in the autolysis of fruiting bodies. It is known that in the structural scaffold
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of the fungal cell β-1,3-glucan with β-1,6-glucan braches is the backbone of the cell
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wall network, the chitin chains are attached to the non-reducing ends of
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β-1,3/1,6-glucans to form the central core of the cell wall architecture27-29. Breaking of
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the backbone of β-1,3/1,6-glucans by β-glucanases could destroy the structural
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scaffold of the fungal cell, while the chitinases may degrade cell walls in synergy with
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the β-1,3-glucanases for pileus autolysis30, 31.
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A significant discovery in this study is that although there are forty-three genes
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annotated as possible glucoside hydrolase genes related to the hydrolysis of cell wall
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β-1,3-glucan, only the following are up-regulated when fruiting bodies cease stipe
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elongation and enter into pileus expansion and autolysis: four known glucoside
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hydrolases genes encode the β-1,3-glucosidase BGL1, the endo-β-1,3-glucanase ENG,
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the exo-β-1,3-glucanase EXG, and the extracellular β-glucosidase12
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unreported putative glucan 1,3-β-glucosidase gene annotated in the NCBI. In a
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previous report, we purified or partially purified these four known β-1,3-glucoside
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hydrolases in larger amounts from an extract of C. cinerea pilei and explored how
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these four β-1,3-glucoside hydrolases act synergistically to completely degrade the 15
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β-1,3-glucan backbone of the C. cinerea cell wall during fruiting body autolysis
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The endo-β-1,3-glucanase ENG hydrolyzes internal glycosidic bonds at glucans to
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generate β-1,3-oligosaccharides of various lengths, exo-β-1,3-glucanase EXG cleaves
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the longer-chain β-1,3-oligosaccharides into short-chain disaccharides, laminaribiose
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and gentiobiose, β-1,3-glucosidase further hydrolyzes laminaribiose to glucose, and,
329
with extensive hydrolytic activity, the extracellular β-glucosidase hydrolyzes the
330
resulting gentiobiose or other oligosaccharides. The unreported putative glucan
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1,3-β-glucosidase may enhance the action of the above known four glucoside
332
hydrolases. It appears that expression of these five glucoside hydrolases is enough to
333
match the requirements for pileus autolysis. Other glucanase genes present in the C.
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cinerea genome may have other physiological roles. To our knowledge, there is no
335
previous report indicating that certain glucanases are specifically involved in the
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autolysis of mature C. cinerea fruiting bodies. This study first explores how the
337
expression of a series of glucanases are controlled. Because these five glucanases
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genes are simultaneously highly expressed in the pileus of fruiting bodies and
339
simultaneously up-regulated when fruiting bodies expand and autolyze, but were
340
simultaneously down-regulated in the non-autolysis mutant, we propose that
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expression of all of these genes are controlled by one regulation system. It would be
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difficult to prove these genes function in vivo by disrupting or silencing of one of
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these glucanase genes, because the autolysis of pileus involve five related glucoside
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hydrolases. The mechanism of the common regulation system for expression of these
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five relative glucoside hydrolases will be elucidated in the future. 16
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As an important food and nutrition product, the commercial value of mushrooms is
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seriously limited due to autolysis postharvest. Like C. cinerea, Volvaria volvacea
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fruiting bodies rapidly autolyse and liquefy postharvest 1. L. edodes fruiting bodies
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exhibit obvious browning and senescence, with lentinan gradually hydrolyzed after
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harvest
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elevation of glucanase activity and up-regulation in glucanase expression during
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fruiting body maturation, but also showed that a non-autolytic mutant has potential
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commercialization prospects. The findings from this study provide insights for the
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selective breeding of non-autolytic mushroom species to expand shelf life and reduce
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the cost of sales.
2,3
. The present study not only revealed the cause of autolysis, namely, an
356 357
Acknowledgments
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This work was supported by National Natural Science Foundation of China (No.
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31170028), the program of Natural Science Research of Jiangsu Higher Education
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Institutions of China (Grant No. 14KJB180013), and NSF of Jiangsu Province of
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China (Project BK20140918).
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Supporting Information description
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Supporting Information Available:
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Table S1 The proteins were annotated as glucanases in the C. cinerea okayama7 #130
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genome.
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Table S2 qPCR primers used in this study. 17
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Page 18 of 29
This material is available free of charge via the Internet at http://pubs.acs.org.
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Figure captions
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Fig. 1. Pileus autolysis of the wild-type (A) and mutant (B) stains of C. cinerea. 1, the
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pileus remained unexpanded (0°); 2, the pileus expanded to approximately 90°; 3, the
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pileus fully expanded to 180°; and 4, 12h after the pileus fully expanded to 180°.
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Fig. 2. DIC images of the pileus of the wild-type (A) and mutant (B) stains of C.
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cinerea 12 h after the pileus fully expanded to 180°. Arrow indicates the basidiospore.
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Fig. 3. The glucanase (A), chitinase (B) and peptidase (C) activities during fruiting
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body maturation in the unexpanded pileus (
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and the fully expanded pileus (
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were obtained at least by three independent experiments with three replicates.
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Statistical tests for significance were done on logtransformed data by oneway
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ANOVA. Values marked with different letters are significantly different, and ones
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with the same letter are not significantly different within the same graph (P